Monoclonal antibodies to antigens expressed by hematopoietic facilitatory cells

ABSTRACT

The present invention relates to monoclonal antibodies (MAb) to hematopoietic facilitatory cells (FC). In particular, it relates to MAb against antigens expressed by murine FC, methods of generating the antibodies, and methods of using the same. MAb directed to markers that are expressed specifically or at higher levels by FC than by most other bone marrow cells have a wide range of applications, including but not limited to, rapid isolation of FC, identification of FC in a donor cell preparation, and molecular cloning of the genes encoding the corresponding target antigens.

INTRODUCTION

[0001] The present invention relates to monoclonal antibodies (MAb) tohematopoietic facilitatory cells (FC). In particular, it relates to MAbagainst antigens expressed by murine FC, methods of generating theantibodies, and methods of using the same. MAb directed to markers thatare expressed specifically or at higher levels by FC than by most otherbone marrow cells have a wide range of applications, including but notlimited to, rapid isolation of FC, identification of FC in a donor cellpreparation, and molecular cloning of the genes encoding thecorresponding target antigens.

BACKGROUND OF THE INVENTION

[0002] A major goal in solid organ transplantation is the engraftment ofthe donor organ without a graft rejection immune response generated bythe recipient, while preserving the immunocompetence of the recipientagainst other foreign antigens. Typically, nonspecific immunosuppressiveagents such as cyclosporine, methotrexate, steroids and FK506 are usedto prevent host rejection responses. They must be administered on adaily basis and if stopped, graft rejection usually results. However,nonspecific immunosuppressive agents function by suppressing all aspectsof the immune response, thereby greatly increasing a recipient'ssusceptibility to infections and diseases, including cancer.

[0003] Furthermore, despite the use of immunosuppressive agents, graftrejection still remains a major source of morbidity and mortality inhuman organ transplantation. Only 50% of heart transplants survive 5years and 20% of kidney transplants survive 10 years. (See Powles, 1980,Lancet, p. 327; Ramsay, 1982, New Engl. J. Med., p. 392). Most humantransplants fail within 10 years without permanent acceptance. It wouldtherefore be a major advance if tolerance can be induced in therecipient.

[0004] The only known clinical condition in which complete systemicdonor-specific transplantation tolerance occurs reliably andreproducibly is when chimerism is created through bone marrowtransplantation. (See Qin et al., 1989, J. Exp. Med. 169:779; Sykes etal., 1988, Immunol. Today 9:23; Sharabi et al., 1989, J. Exp. Med.169:493). This has been achieved in neonatal and adult animal models aswell as in humans by total lymphoid irradiation of a recipient followedby bone marrow transplantation with donor cells. The widespreadapplication of bone marrow transplantation to areas outside ofmalignancy has been limited by graft-versus-host disease (GVHD). Thesuccess rate of bone marrow transplantation is, in part, dependent onthe ability to closely match the major histocompatibility complex (MHC)of the donor cells with that of the recipient cells. The MHC is a genecomplex that encodes a large array of individually unique glycoproteinsexpressed on the surface of both donor and host cells that are the majortargets of transplantation rejection immune responses. In the human, theMHC is referred to as HLA. When HLA identity is achieved by matching apatient with a family member such as a sibling, the probability of asuccessful outcome is relatively high, although GVHD is still notcompletely eliminated. The incidence and severity of GVHD are directlycorrelated with degree of genetic disparity. In fact, only one or twoantigen mismatch is acceptable because GVHD is very severe in cases ofgreater disparities. When allogeneic bone marrow transplantation isperformed between two MHC-mismatched individuals of the same species,common complications involve failure of engraftment, poorimmunocompetence and a high incidence of GVHD.

[0005] GVHD is a potentially lethal complication in bone marrowtransplantation, which occurs in about 35-50% of recipients of untreatedHLA-identical marrow grafts (Martin et al., 1985, Blood 66:664) and upto 80% of recipients of HLA-mismatched marrow. Unfortunately, only 30%of patients generally have a suitably matched HLA-identical familymember donor, and thus most patients are either excluded from beingconsidered for bone marrow transplantation, or if they are transplantedmust tolerate a high risk of GVHD. GVHD results from the ability ofimmunocompetent mature immune cells (mainly T cells, but some B cellsand natural killer cells) in the donor graft to recognize host tissueantigens as foreign and invoke an adverse immunologic reaction.Although, mixed allogeneic reconstitution, in which a mixture of donorand recipient marrow is transplanted, results in improvedimmunocompetence and increased resistance to GVHD, successfulengraftment is still not consistently achieved and GVHD still oftenoccurs.

[0006] Recent studies in bone marrow transplantation suggest that themajor cause of GVHD are T-cells, as the removal of T cells from thedonor cell preparation was associated with a reduction in the incidenceof GVHD. (Vallera et al., 1989, Transplant, 47:751; Rayfield, 1984, Eur.J. Immunol., P. 308; Vallera, 1982, J. Immunol., 128:871; Martin andKorngold, 1978, J. Exp. Med., p 1687; Prentice, 1984, Lancet P. 472).After T-cells were implicated to be the predominant mediator of GVHD inanimal models, aggressive protocols for T-cell depletion (TCD) of humandonor bone marrow were instituted. Although the incidence of GVHD wasdecreased dramatically, TCD was accompanied by a significant increase inthe failure of engraftment, indicating that T cells might also play apositive role in bone marrow engraftment. (Soderling, J. Immunol., 1985,135:941; Vallera, 1982, Transplant. 33:243; Pierce, 1989, Transplant.,p. 289). The increase in failure of engraftment in human recipientsranged from about 5-70% of total patients and was related to the degreeof MHC disparity between the donor and recipient (Blazar, 1987, UCLASymp., p. 382; Filipovich, 1987, Transplant., p. 62; Martin et al.,1985, Blood 66:664; Martin et al., 1988, Adv. Immunol. 40:379). Patientswith failed engraftment usually die even if a second bone marrowtransplant is performed. Consequently, most transplant institutions inthe United States have abandoned TCD of donor bone marrow and, thus,must tolerate a high level of GVHD which leads to significant morbidityand mortality. Thus, the application of bone marrow transplantation as aform of treatment is limited only to settings where the potential ofGVHD is clearly outweighed by the potential benefit. It was thereforeanticipated that the administration of purified bone marrow stem cellswould optimize engraftment and avoid GVHD. However, recent studies haveshown that purified bone marrow stem cells only engraft in geneticallyidentical, but not in genetically disparate recipients.

[0007] The implication that T cells might participate in both harmfulGVHD reactions and helpful engraftment facilitation was an enigma thatexisted for a long time in the scientific community. Investigators beganto search for the possible existence of a bone marrow component whichcould facilitate bone marrow engraftment but was removed during TCD.Identification and purification of this facilitating component wouldpotentially allow the design of transplant protocols to selectivelyprevent GVHD, while preserving the cells that can enhance engraftment.

[0008] Although most investigators speculated that the facilitatingcomponent was a hematopoietic cell distinct from the hematopoietic stemcells, such a component had never been identified or characterized untilrecently. In fact, all evidence pointed towards the involvement of someform of T cells. It was recently discovered that a cell populationreferred to as FC facilitates engraftment of hematopoietic stem cells ina recipient without producing GVHD, and this cell expresses severalmarkers shared by other leukocytes. The identification of specificmarkers expressed by FC would greatly assist the rapid isolation of thiscell type.

SUMMARY OF THE INVENTION

[0009] The present invention relates to MAb directed to antigensexpressed by murine FC, methods of generating the antibodies and methodsof using the same to isolate FC.

[0010] The invention is based, in part, on the Applicants' discoverythat FC play a critical role in promoting the ability of donorhematopoietic stem cells to engraft in a lethally-irradiated allogeneicor xenogeneic recipient. Although murine FC are morphologically distinctfrom all other known cell types and they have been shown to expressThy-1, CD2, CD3, CD5, CD8, CD45, CD45R and MHC class II (in the low tointermediate range as compared to B cells and dendritic cells), thesemarkers individually do not readily distinguish the FC from other bonemarrow cells. Therefore, the isolation and enrichment of FC currentlyemploy a cumbersome and time-consuming multiple step procedure involvingpositive and negative selection. In order to develop a method for rapididentification of FC in a cell mixture and their subsequent isolationtherefrom, MAb may be produced to antigens specifically or moreselectively expressed by FC than by other cells, assuming such antigensexist.

[0011] The generation of MAb requires the use of FC as immunogens, butsince FC are present in natural tissue sources at low quantities(approximately 0.05%), it is practically difficult to obtain a highyield of an enriched population of FC for use in immunization. Whilewhole bone marrow preparation with little or no enrichment for FC may beused as immunogens, it is unlikely that MAb can be raised to FC markerssince other bone-marrow cells are present in much higher numbers andexpress other highly immunogenic antigens which may dominate theantibody responses to the FC-associated molecules.

[0012] In an effort to generate MAb to FC, it is recognized that FC mayshare certain cell surface antigens with brain tissue as shown by theability of rabbit-anti-mouse brain (RAMB) antiserum to reduce the levelof donor bone marrow cell engraftment, presumably due to a depletion ofFC. Thus, brain tissue is prepared and used to immunize animals. Cellfusion is performed using spleen cells from immunized animals and theresultant hybridomas are first screened for the secretion of antibodiesin their supernatants. Thereafter, the MAb are further screened fortheir ability to deplete FC activity in vivo, as manifested by mixedallogeneic chimerism in recipients following reconstitution with donorbone marrow cells treated with the antibodies. MAb exhibiting suchactivities in this screening procedure are selected for furthercharacterization.

[0013] The invention is described by way of examples in which mousebrain tissue is prepared and used to immunize rats. After severalimmunizations, the rats are sacrificed and their spleen cells fused withmouse myeloma cells. The resultant hybridomas are first screened fortheir secretion of rat antibodies of IgG or IgM isotypes. The positivehybridomas are further tested by reacting their supernatants with mousedonor (H-2^(k)) bone marrow cells prior to their co-administration withTCD H-2^(b) donor bone marrow into H-2^(b) recipients. In this model,untreated allogeneic donor bone marrow cells give rise to fully (100%)allogeneic chimeras, whereas RAMB or anti-Thy-1 antibody-treated donorcells produce low levels of mixed allogeneic chimerism, if any, inrecipients, presumably due to the diminution of FC in the donor cellpreparation. Three MAb, designated R7.6.2, R340.3.1 and R373.6.3 arecapable of depleting FC, producing mixed allogeneic chimeras. A widevariety of uses for MAb to antigens expressed by FC are encompassed bythe invention described herein, including but not limited to, theidentification of FC in a donor cell preparation, the isolation andenrichment of FC from a cell mixture, and the molecular cloning of thecorresponding target antigens.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1A Untreated donor bone marrow cells produce and 1B fullyallogeneic chimeras. Only allogeneic cells (H-2^(k)) are detected.

[0015]FIG. 2A RAMB-treated donor bone marrow cells produce and 2B mixedallogeneic chimeras. Both syngeneic and allogeneic cells are detected.

[0016]FIG. 3A Anti-Thy1.2-treated donor bone marrow cells and 3B producemixed allogeneic chimeras. Both syngeneic and allogeneic cells aredetected.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention relates to MAb to antigens expressed bymurine FC, methods of generating such antibodies and uses of such MAb.Although the specific procedures and methods described herein areexemplified using murine brain tissue for inducing rat MAb against mouseFC, they are merely illustrative for the practice of the invention.Analogous procedures and techniques are equally applicable to a varietyof animal hosts immunized against brain tissue, partially purified FC orFC antigens for producing MAb against FC markers, including thatexpressed by human FC.

[0018] Preparation of Immunogens

[0019] In order to generate MAb to antigens selectively expressed by FC,there are two major hindrances that must first be overcome. The firstrelates to the low quantities of FC in natural tissues and thus theyneed to be enriched to sufficient quantities and in relatively pure formfor use as immunogens. It is estimated that it would require 4000 hoursof cell sorting to obtain sufficient numbers of purified FC from bonemarrow for use in immunization of one animal, if FC are purified to >95%purity.

[0020] Although the activity of FC allows for the use of these cells inrelatively small numbers when enriched, it is preferred that they beenriched to >50% for use as immunogens. FC may be isolated from anytissues where they reside, using a variety of separation methods. Inaccordance with this aspect of the invention, human FC may be isolatedfrom bone marrow. Procedures involving repetitive density gradientcentrifugation, positive selection, negative selection, or a combinationthereof may be used. For example, the human FC may be prepared bysubjecting bone marrow aspirates to “FICOLL HYPAQUE” centrifugation.Positive selection does not necessarily require the use of antibodiesthat recognize FC-specific determinants. For example, B cells andmonocytes may be depleted first from the FC-containing fraction afterdensity gradient centrifugation, plastic adhesion, and Fc receptorpanning, then an antibody to MHC-Class II antigen can be used topositively select for FC. Negative selection includes modifications ofthe protocol disclosed herein. For example, a FC-containing cellpreparation may be reacted with one or more antibodies directed at cellsurface antigens not expressed by FC for the removal of non-FC.Antibodies to a number of T cell, B cell, monocyte, and granulocytemarkers may be used. Examples of such antibodies include anti-CD4 andanti-TCR specific for T cells; anti-CD12, anti-CD19 and anti-CD20specific for B cells; anti-CD14 specific for monocytes; and anti-CD16and anti-CD56 specific for natural killer cells. These antibodies may beapplied in any combination repeatedly or in a sequential manner for theenrichment of FC. Upon binding to the antibodies, the cells may beremoved by adsorption to a solid surface coated with an anti-mouseantibody, as the majority of monoclonal antibodies directed at humancell surface markers are of mouse origin, or if the antibodies areconjugated with biotin, the antibody-bound cells can be removed by anavidin or streptavidin-coated surface; or if the antibodies areconjugated to magnetic beads, the cells expressing antigens recognizedby the antibodies can be removed in a magnetic field (Harlow and Lane,1988, Antibody: A Laboratory Manual, Cold Spring Harbor).

[0021] Current methods for FC enrichment require a series of positiveand negative selection steps, therefore other sources of FC-associatedantigens may be used. For example, brain tissue appears to contain thesame or cross-reactive antigens as that expressed by FC, and may beprepared for use as immunogens for the production of anti-human FC MAb.Brain tissue from any species may be obtained and prepared for use inimmunization in the same manner as described in Section 6.1.2, infra,except that large tissue should be cut into small sections prior tohomogenization.

[0022] The second hindrance relates to an efficient method fordifferential screening of the specific antibodies desired, i.e., toselect for antibodies that are directed to FC but less so to other bloodcells. For the purpose of the instant application, FC are defined asbone marrow-derived cells of about 8-10 microns in diameter, capable ofenhancing stem cell engraftment, and which express Thy-1, CD3, CD8,CD45, CD45R, MHC class II (low to intermediate levels), but lack othermarkers such as CD4, CD5, CD14, CD16, CD19, CD20, CD56, γδ-TCR andαβ-TCR. MAb may be screened by binding assays in which the antibodiesbind to FC but not or to a lesser degree to other bone marrow cellsincluding stem cells, T cells, B cells, macrophages, monocytes,granulocytes, red blood cells and platelets. Antibody staining may bedetermined by flow cytometry or any other detection methods known in theart. Alternatively, antibodies may be screened for their ability todeplete FC function such as in an in vivo engraftment assay described inSection 6, infra.

[0023] Antibody Production

[0024] Various methods may be used to produce polyclonal and monoclonalantibodies that recognize novel antigenic markers expressed by FC. Anyprocedure known in the art may be used for the production of antibodiesto these cells. For the production of antibodies, various host animalscan be immunized by injection with viable, purified or partiallypurified FC or brain tissue, fixed cells or membrane preparations,including, but not limited to, those of rabbits, hamsters, mice, rats,etc. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum.

[0025] MAb which are substantially homogeneous antibodies to singleantigenic epitopes on FC may be prepared by using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique originally described by Kohler and Milstein (1975, Nature 256,495-497), the more recent human B-cell hybridoma technique (Kosbor etal., 1983, Immunology Today 4:72; Cote et al., 1983, Proc. Natl. Acad.Sci. USA 80:2026-2030) and the EBV-hybridoma technique (Cole et al.,1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). MAb can be screened differentially by selective binding to FC,but not to mature macrophages, granulocyte, monocytes, T cells, B cells,stem cells and dendritic cells, and/or by inhibition of FC activity.

[0026] Antibody fragments which contain the binding site of the moleculemay be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragments which can beproduced by pepsin digestion of the antibody molecule and the Fabfragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments.

[0027] A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine or rat MAb and a human immunoglobulinconstant region. Techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851;Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985,Nature, 314:452-454) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.This approach is particularly useful if the antibodies are administeredinto humans. Chimeric antibodies present less xenogeneic epitopes ininducing an anti-rodent Ig response when injected in man.

[0028] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-425; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adaptedto produce FC-reactive single chain antibodies. Single chain antibodiesare formed by linking the heavy and light chain fragment of the Fvregion via an amino acid bridge, resulting in a single chainpolypeptide.

[0029] Additionally, the whole antibody molecule or its Fab, F(ab′)₂ orF_(v) fragment may be conjugated to any of a variety of compoundsincluding, but not limited to, signal generating compounds such as afluorochrome, radio-isotope, a chromophore, an enzyme, achemiluminescent or bioluminescent molecule, etc. Alternatively, thewhole antibody or its Fab, F(ab′)₂ or F_(v) fragment may be conjugatedto a cytokine which may enhance or inhibit the biological activity ofFC; or to toxins so that FC which express the corresponding antigenswould be selectively killed (Vitetta and Uhr, 1985, Annu Rev. Immunol.3:197). Methods which can be used for conjugating labels, proteins,toxins etc. to antibodies and antibody fragments are well known in theart (See, for example, U.S. Pat. Nos. 4,220,450; 2,235,869; 3,935,074and 3,996,345).

[0030] Uses of Monoclonal Antibodies to Hematopoietic Facilitatory Cells

[0031] A variety of uses of MAb are encompassed by the presentinvention. An antibody exhibiting exquisite specificity for FC in thatit does not bind to T cells, B cells, NK cells, granulocytes,macrophages, monocytes, red blood cells, platelet and stem cells, may beused to isolate FC in a one step affinity cell separation procedure.Antibodies to markers that are selectively expressed by FC, i.e.,certain but not all blood cells also express it, may still be usedeffectively in combination with other methods such as density gradientcentrifugation to substantially reduce the time-consuming and cumbersomeprocedures currently employed for the isolation of FC.

[0032] For the practice of this aspect of the invention, a MAb may beconjugated to fluorochromes and used to select for FC from a cellmixture by flow cytometry using a fluorescence activated cell sorter ormay be conjugated to biotin for use in biotin-avidin orbiotin-streptavidin separations. In the latter method, avidin orstreptavidin is bound to a solid support such as affinity column matrixor plastic surfaces. In addition, antibodies may be coated with magneticbeads, reacted with a cell mixture, and the antibody-bound FC removed bya magnetic field. Furthermore, such MAb may be conjugated to an enzymefor use in immunohistochemistry. For example, certain disorders may beinduced or sustained by an aberrant function of FC, and detection of thelevel of FC in tissue sections may be of diagnostic value.

[0033] Additionally, MAb directed to FC markers may be used to isolateand identify the genes encoding such molecules. Antibodies may be usedfor screening expression libraries made from FC for the molecularcloning of the coding sequences (Seed and Aruffo, 1987, Proc. Natl.Acad. Sci. USA 84:3365-3369).

EXAMPLE: Generation of Monoclonal Antibodies to Murine HematopoieticFacilitatory Cells

[0034] Materials and Methods

[0035] Animals

[0036] Six to eight week old male C57BL/10SnJ (B10), and B10.BR/SgSn(B10.BR) mice were purchased from the Jackson Laboratory (Bar Harbor,Me.). Four to eight week old male Fischer 344 (F344) male rats werepurchased from Harlan Sprague Dawley, Inc. (Indianapolis, Ind.). Animalswere housed in a specific pathogen-free facility at the BiomedicalScience Tower at the University of Pittsburgh.

[0037] Immunization and Cell Fusion

[0038] Mouse brain tissue was obtained enbloc from calvarium. Braintissue was placed in 1 ml PBS for each 1 cm³ of brain tissue, which washomogenized in a glass homogenizer. Brain emulsion was then mixed withComplete Freund's Adjuvant at 1:1 ratio prior to animal injection. 0.4ml of homogenized mouse brain was emulsified in adjuvant. Rat injectionswere done subcutaneously every 2 weeks for a total of four injections.Three days following the fourth injection, the peripheral blood wastested for antibody production. The animals exhibiting the strongestactivities were then selected to be utilized for hybridoma fusion. Theselected animals were then given an additional injection of brain/PBSmixture subcutaneously in the absence of complete Freund's adjuvant.This injection was generally about 5 to 6 days after the fourthinjection. Two days following this fifth injection, the spleens of theanimals were harvested and fused with HGPRT myeloma cells (P3.653) usingpolyethylene glycol (Kohler and Milstein, 1975, Nature 256:495). Thecells were then distributed in microwell plates and grown in HAT medium(RPMI-1640 supplemented with 10% fetal bovine serum, 1% Pen/Strep, 1%L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate and HAT).The unfused myeloma cells died because of their lack of HGPRT to use thesalvage pathway. The unfused spleen cells also died because they wereunable to grow in vitro. The fused cells (hybridomas) grew in themicrowells and their culture supernatants were first tested for theproduction of rat antibodies.

[0039] The culture supernatant was screened for the presence of ratanti-mouse antibodies by incubating 10⁶ mouse bone marrow cells in smallflow tubes with rat serum to block non-specific rat antibody staining.20-30 μl of hybridoma culture supernatant was then added to each tube ofbone marrow, two separate tubes were tested for each supernatant—one toscreen for IgG production and the other for IgM production. Following a45 min. incubation at 4° C., cells were washed twice at 1000 rpm×10 min.and the media decanted. Pre-titered goat-anti-rat IgG-FITC was added tothe first tube for each culture supernatant, and anti-rat IgM-PE wasadded to the second tube. After 45 minute incubation at 4° C., the cellswere washed twice and fixed in 0.4 ml of 1% paraformaldehyde forsubsequent flow cytometric analysis. The controls included samples withcells alone, IgG alone, IgM alone as assessments of background stainingand negative controls; and RAMB, Lyt2-FITC, and unlabelled rat IgG andIgM MAb against known mouse antigens as positive controls. Hybridomaswere selected for the production of rat anti-mouse antibodies whichcross reacted with distinct populations of mouse bone marrow. Thepositive wells were further screened for antibodies directed to FC in anin vivo assay. The selected hybridomas were cloned by limiting dilution.The cloned hybridomas were injected into pristane-primed nude mice forthe production of ascites.

[0040] Preparation of Mixed Allogeneic Chimeras

[0041] In order to screen and select for MAb directed to FC, apreparation of donor bone marrow cells was reacted with hybridomasupernatants prior to their injection into allogeneic mouse recipients.Mixed allogeneic chimerism in the recipients was used as an indicator ofthe presence of MAb capable of depleting FC function. To prepare mixedchimeras, bone marrow from the long bones of syngeneic (B10) mice andallogeneic (B10.BR) mice were harvested. The mice were euthanized withCO₂ narcosis, prepared with 70% alcohol, and the long hind bone (femoraand tibia) removed. The marrow was flushed from the bones using medium199 (Gibco Laboratories Life Technology, Inc., Grand Island, N.Y.)supplemented with 50 μl/ml of gentamicin using a 22-gauge needle. Themedium mixture (MEM) was used to mechanically resuspend the bone marrowby gentle aspiration through an 18-gauge needle and the suspensionfiltered through sterile nylon mesh gauze. The cells were then pelletedat 1000 rpm for 10 minutes, resuspended in MEM, and counted. In standardallogeneic reconstitution, RAMB was used for T-cell depletion ofsyngeneic B10 bone marrow (1:40 or appropriate dilution at 10⁸ cells/mlat 4° C. for 30 minutes). RAMB was prepared in the same manner as thatdescribed for immunization of rats with mouse brain in Section 6.1.2,supra, except that mouse brain was used to immunize rabbits. Theallogeneic B10.BR bone marrow cells were either untreated,RAMB-depleted, anti-Thy1.2 depleted or hybridoma supernatant treated.10×10⁶ donor bone marrow cells were pelleted and antibodies added 1:10in 1 ml. The media were prewarmed to 37° C. so that the antibodyincubation was performed at 37° C. for 30 minutes. Cells were thenwashed in MEM, spun at 1000 rpm for 10 minutes and resuspended in guineapig complement at 37° C. for 30 minutes (Gibco Laboratories LifeTechnology, Inc., Grand Island, N.Y.). Cells were washed twice, countedand resuspended in MEM at the appropriate concentration to allowinjection of 1 ml of total volume per animal. The RAMB-treated syngeneiccells were injected at 5×10⁶/animal, whereas the allogeneic cells weregiven at 15×10⁶/animal within 4-6 hours after irradiation of recipientanimals at 9.5 Gy. Cell injections were via the lateral tail veins usinga 27-gauge needle.

[0042] Characterization of Chimeras by Flow Cytometry

[0043] Recipients were characterized for engraftment with syngeneic andallogeneic donor lymphoid elements using flow cytometry to determine thepercentage of peripheral blood leukocytes (PBL) bearing MHC Class I(H-2^(b) or H-2^(k)) surface markers. Briefly, peripheral blood wascollected into heparinized plastic serum vials. After thorough mixing,the suspension was layered over 1.5 ml of room temperature lymphocyteseparation medium (LSM) (Organon Technical, Kensington, Md.) andcentrifuged at 20° C. at 1700 rpm for 30 minutes. The lymphocyte layerwas aspirated from the saline-LSM interface and washed with medium. Redblood cells were ACK-lysed (ammonium chloride/potassium carbonate lysingbuffer) and the remaining cells stained with appropriate MAb for 30minutes at 4° C. and counterstained with sandwich when required.Analyses of splenic and thymic lymphoid cells were performed using afluorescence activated cell sorter (FACS) (FACS II Becton Dickinson andCompany, Mountain View, Calif.).

[0044] Results

[0045] The experiments described in the following sections utilized amixed chimera model in which recipient animals were lethally-irradiatedand transplanted with fixed doses of allogeneic donor cells andsyngeneic donor cells. The percentage of allogeneic chimerism, i.e., thelevel of mixed chimerism was used as a read-out of FC activity inpromoting donor cell engraftment.

[0046] It was previously reported that RAMB and complement treatment ofa bone marrow preparation negatively affected its ability to engraft ina recipient. Recently, a bone marrow cell population referred to as FChas been identified, which greatly enhanced hematopoietic stem cellengraftment. In allogeneic bone marrow transplantation, Sca-1⁺ purifiedhematopoietic stem cells alone were not able to engraft unless FC wereco-administered. Furthermore, FC did not possess stem cell activity.Since RAMB appeared to deplete FC and RAMB was an antiserum raisedagainst mouse brain, it was possible that mouse FC shared certain commonor cross-reactive antigens with mouse brain tissues. Thus, mouse braintissues were obtained, homogenized and used as immunogens in rats forthe production of MAb against markers expressed by mouse FC.

[0047] After several immunizations with mouse brain tissues, rats weresacrificed and their spleen cells fused with HGPRT myeloma cells bypolyethylene glycol. The resulting hybridoma cells were selected in HATmedium and their culture supernatants tested for their ability to reducethe level of donor bone marrow engraftment in allogeneic recipients asan indication of the presence of antibodies capable of eliminating FC.

[0048] The antibody screening procedure utilized an established mixedallogeneic chimerism model in which mouse recipients receivedTCD-syngeneic bone marrow cells plus allogeneic bone marrow cellstreated with various antibodies. The level of allogeneic chimerism inthe recipients was determined by the use of anti-MHC class I antibodies,and it was used as a an indication of the effects of antibodies on FCfunction. For example, an untreated allogeneic donor bone marrowpreparation led to fully allogeneic recipients, i.e. primarily H-2^(k)allogeneic cells, with few syngeneic (H-2^(b)) cells were detected inthe recipients (FIG. 1A and 1B). On the other hand, allogeneic donorcells treated with RAMB (FIG. 2A and 2B) or anti-Thy1.2 (FIG. 3A and 3B)antibody led to mixed allogeneic chimerism, indicating that thesereagents depleted FC which were needed to promote allogeneic stem cellengraftment.

[0049] As compared to these controls, donor bone marrow cells weretreated with hybridoma supernatants and subsequently transplanted intoallogeneic recipients to select for antibodies that would produce mixedallogeneic chimerism similar to the results obtained with RAMB oranti-Thy1.2 treatment. Antibodies that did not reduce the level of fullallogeneic chimerism were discarded since they were not able to removeFC.

[0050] Out of the numerous hybridomas generated and the ones tested inthe aforementioned assay, three hybridoma cell lines designated R7.6.2(IgG2a), R340.3.1 (IgM) and R373.6.3 (IgM) were selected for furtherstudies. These cell lines produced antibodies which were directed to FCmarkers as evidenced by their ability to cause mixed allogeneicchimerism in recipients transplanted with donor cells treated with them,while untreated donor cells produced fully allogeneic chimeras (TableI). These results indicated that the three MAb were directed to FC,capable of depleting FC in the donor cell preparation and in turn,causing a diminution in the ability of the stem cells to engraft. Out ofa total of greater than 150 hybridomas screened, only three clonesproduced antibodies that bound preferentially to FC. TABLE I MONOCLONALANTIBODIES DIRECTED TO FC CLONE # animal H-2^(b) (syngeneic) H-2^(k)(allogeneic) R7.6.2 #959 18.90 79.12 #961 12.12 86.98 #962 12.48 91.18#964 10.66 93.26 #966 27.35 70.34 #967 12.58 88.18 R340.3.1 #965 40.0667.98 #968 9.2 98.24 R373.6.3 #773 85.54 13.91 #774 97.28 0.54 #77535.89 57.57 #974 99.76 0.38 RAMB #272 50.78 48.02 Depletion #273 49.2246.72 #274 49.34 44.04 #275 97.54 5.74 Anti-Thy1.2 #969 63.34 33.56Depletion #971 64.52 32.90 B10 Control 95.52 0.56 B10.BR 0.96 99.24Control

[0051] Deposit of Cell Line

[0052] The following hybridoma cell lines were deposited with theAmerican Type Culture Collection, Rockville, Maryland and assigned thefollowing accession numbers: Hybridoma ATCC Accession Number R7.6.2R340.3.1 R373.6.3 HB11507

[0053] The present invention is not to be limited in scope by theexemplified embodiments which are intended as illustrations of singleaspects of the invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

[0054] All publications cited herein are incorporated by reference intheir entirety. selecting fused cells which secrete antibodies capableof depleting facilitatory cells.

What is claimed is:
 1. A monoclonal antibody, the antigen-binding regionof which competitively inhibits the immunospecific binding of monoclonalantibody produced by hybridoma R7.6.2 having an ATCC accession number ₁₃₁₃ ₁₃ ₁₃ to its target epitope.
 2. A monoclonal antibody, theantigen-binding region of which competitively inhibits theimmunospecific binding of monoclonal antibody produced by hybridomaR340.3.1 having an ATCC accession number ₁₃ ₁₃ ₁₃ ₁₃ to its targetepitope.
 3. A monoclonal antibody, the antigen-binding region of whichcompetitively inhibits the immunospecific binding of monoclonal antibodyproduced by hybridoma R373.6.3 having an ATCC accession number HB11507to its target epitope.
 4. Monoclonal antibody produced by hybridomaR.7.6.2 as deposited with the ATCC.
 5. Monoclonal antibody produced byhybridoma R340.3.1 as deposited with the ATCC.
 6. Monoclonal antibodyproduced by hybridoma R373.6.3 as deposited with the ATCC.
 7. A methodof generating monoclonal antibodies to antigens expressed byhematopoietic facilitatory cells comprising immunizing an animal withhomogenized brain tissue, fusing spleen cells from the animal with afusion partner cell line, and